Genomic evidence reveals three W-autosome fusions in Heliconius butterflies.

Sex chromosomes are evolutionarily labile in many animals and sometimes fuse with autosomes, creating so-called neo-sex chromosomes. Fusions between sex chromosomes and autosomes have been proposed to reduce sexual conflict and to promote adaptation and reproductive isolation among species. Recently, advances in genomics have fuelled the discovery of such fusions across the tree of life. Here, we discovered multiple fusions leading to neo-sex chromosomes in the sapho subclade of the classical adaptive radiation of Heliconius butterflies. Heliconius butterflies generally have 21 chromosomes with very high synteny. However, the five Heliconius species in the sapho subclade show large variation in chromosome number ranging from 21 to 60. We find that the W chromosome is fused with chromosome 4 in all of them. Two sister species pairs show subsequent fusions between the W and chromosomes 9 or 14, respectively. These fusions between autosomes and sex chromosomes make Heliconius butterflies an ideal system for studying the role of neo-sex chromosomes in adaptive radiations and the degeneration of sex chromosomes over time. Our findings emphasize the capability of short-read resequencing to detect genomic signatures of fusion events between sex chromosomes and autosomes even when sex chromosomes are not explicitly assembled.

Optix and cortex/ivory/mir-193 again: the repeated use of two mimicry hotspot loci.

The extent to which evolution is repeatable has been a debated topic among evolutionary biologists. Although rewinding the tape of life perhaps would not lead to the same outcome every time, repeated evolution of analogous genes for similar functions has been extensively reported. Wing phenotypes of butterflies and moths have provided a wealth of examples of gene re-use, with certain 'hotspot loci' controlling wing patterns across diverse taxa. Here, we present an example of convergent evolution in the molecular genetic basis of Batesian wing mimicry in two Hypolimnas butterfly species. We show that mimicry is controlled by variation near cortex/ivory/mir-193, a known butterfly hotspot locus. By dissecting the genetic architecture of mimicry in Hypolimnas misippus and Hypolimnas bolina, we present evidence that distinct non-coding regions control the development of white pattern elements in the forewing and hindwing of the two species, suggesting independent evolution, and that no structural variation is found at the locus. Finally, we also show that orange coloration in H. bolina is associated with optix, a well-known patterning gene. Overall, our study once again implicates variation near the hotspot loci cortex/ivory/mir-193 and optix in butterfly wing mimicry and thereby highlights the repeatability of adaptive evolution.

Rapid Adaptation and Interspecific Introgression in the North American Crop Pest Helicoverpa zea.

Insect crop pests threaten global food security. This threat is amplified through the spread of nonnative species and through adaptation of native pests to control measures. Adaptations such as pesticide resistance can result from selection on variation within a population, or through gene flow from another population. We investigate these processes in an economically important noctuid crop pest, Helicoverpa zea, which has evolved resistance to a wide range of pesticides. Its sister species Helicoverpa armigera, first detected as an invasive species in Brazil in 2013, introduced the pyrethroid-resistance gene CYP337B3 to South American H. zea via adaptive introgression. To understand whether this could contribute to pesticide resistance in North America, we sequenced 237 H. zea genomes across 10 sample sites. We report H. armigera introgression into the North American H. zea population. Two individuals sampled in Texas in 2019 carry H. armigera haplotypes in a 4 Mbp region containing CYP337B3. Next, we identify signatures of selection in the panmictic population of nonadmixed H. zea, identifying a selective sweep at a second cytochrome P450 gene: CYP333B3. We estimate that its derived allele conferred a ∼5% fitness advantage and show that this estimate explains independently observed rare nonsynonymous CYP333B3 mutations approaching fixation over a ∼20-year period. We also detect putative signatures of selection at a kinesin gene associated with Bt resistance. Overall, we document two mechanisms of rapid adaptation: the introduction of fitness-enhancing alleles through interspecific introgression, and selection on intraspecific variation.

Selection drives divergence of eye morphology in sympatric Heliconius butterflies.

When populations experience different sensory conditions, natural selection may favor sensory system divergence, affecting peripheral structures and/or downstream neural pathways. We characterized the outer eye morphology of sympatric Heliconius butterflies from different forest types and their first-generation reciprocal hybrids to test for adaptive visual system divergence and hybrid disruption. In Panama, Heliconius cydno occurs in closed forests, whereas Heliconius melpomene resides at the forest edge. Among wild individuals, H. cydno has larger eyes than H. melpomene, and there are heritable, habitat-associated differences in the visual brain structures that exceed neutral divergence expectations. Notably, hybrids have intermediate neural phenotypes, suggesting disruption. To test for similar effects in the visual periphery, we reared both species and their hybrids in common garden conditions. We confirm that H. cydno has larger eyes and provide new evidence that this is driven by selection. Hybrid eye morphology is more H. melpomene-like despite body size being intermediate, contrasting with neural trait intermediacy. Overall, our results suggest that eye morphology differences between H. cydno and H. melpomene are adaptive and that hybrids may suffer fitness costs due to a mismatch between the peripheral visual structures and previously described neural traits that could affect visual performance.

Transposable Element Insertions Are Associated with Batesian Mimicry in the Pantropical Butterfly Hypolimnas misippus.

Hypolimnas misippus is a Batesian mimic of the toxic African Queen butterfly (Danaus chrysippus). Female H. misippus butterflies use two major wing patterning loci (M and A) to imitate three color morphs of D. chrysippus found in different regions of Africa. In this study, we examine the evolution of the M locus and identify it as an example of adaptive atavism. This phenomenon involves a morphological reversion to an ancestral character that results in an adaptive phenotype. We show that H. misippus has re-evolved an ancestral wing pattern present in other Hypolimnas species, repurposing it for Batesian mimicry of a D. chrysippus morph. Using haplotagging, a linked-read sequencing technology, and our new analytical tool, Wrath, we discover two large transposable element insertions located at the M locus and establish that these insertions are present in the dominant allele responsible for producing mimetic phenotype. By conducting a comparative analysis involving additional Hypolimnas species, we demonstrate that the dominant allele is derived. This suggests that, in the derived allele, the transposable elements disrupt a cis-regulatory element, leading to the reversion to an ancestral phenotype that is then utilized for Batesian mimicry of a distinct model, a different morph of D. chrysippus. Our findings present a compelling instance of convergent evolution and adaptive atavism, in which the same pattern element has independently evolved multiple times in Hypolimnas butterflies, repeatedly playing a role in Batesian mimicry of diverse model species.

Rapid evolution of novel biotic interactions in the UK Brown Argus butterfly uses genomic variation from across its geographical range.

Understanding the rate and extent to which populations can adapt to novel environments at their ecological margins is fundamental to predicting the persistence of biological communities during ongoing and rapid global change. Recent range expansion in response to climate change in the UK butterfly Aricia agestis is associated with the evolution of novel interactions with a larval food plant, and the loss of its ability to use an ancestral host species. Using ddRAD analysis of 61,210 variable SNPs from 261 females from throughout the UK range of this species, we identify genomic regions at multiple chromosomes that are associated with evolutionary responses, and their association with demographic history and ecological variation. Gene flow appears widespread throughout the range, despite the apparently fragmented nature of the habitats used by this species. Patterns of haplotype variation between selected and neutral genomic regions suggest that evolution associated with climate adaptation is polygenic, resulting from the independent spread of alleles throughout the established range of this species, rather than the colonization of pre-adapted genotypes from coastal populations. These data suggest that rapid responses to climate change do not depend on the availability of pre-adapted genotypes. Instead, the evolution of novel forms of biotic interaction in A. agestis has occurred during range expansion, through the assembly of novel genotypes from alleles from multiple localities.

Colour polymorphism associated with a gene duplication in male wood tiger moths.

Colour is often used as an aposematic warning signal, with predator learning expected to lead to a single colour pattern within a population. However, there are many puzzling cases where aposematic signals are also polymorphic. The wood tiger moth, Arctia plantaginis, displays bright hindwing colours associated with unpalatability, and males have discrete colour morphs which vary in frequency between localities. In Finland, both white and yellow morphs can be found, and these colour morphs also differ in behavioural and life-history traits. Here, we show that male colour is linked to an extra copy of a yellow family gene that is only present in the white morphs. This white-specific duplication, which we name valkea, is highly upregulated during wing development. CRISPR targeting valkea resulted in editing of both valkea and its paralog, yellow-e, and led to the production of yellow wings. We also characterise the pigments responsible for yellow, white, and black colouration, showing that yellow is partly produced by pheomelanins, while black is dopamine-derived eumelanin. Our results add to a growing number of studies on the genetic architecture of complex and seemingly paradoxical polymorphisms, and the role of gene duplications and structural variation in adaptive evolution.

Evolutionary dynamics of genome size and content during the adaptive radiation of Heliconiini butterflies.

Heliconius butterflies, a speciose genus of Müllerian mimics, represent a classic example of an adaptive radiation that includes a range of derived dietary, life history, physiological and neural traits. However, key lineages within the genus, and across the broader Heliconiini tribe, lack genomic resources, limiting our understanding of how adaptive and neutral processes shaped genome evolution during their radiation. Here, we generate highly contiguous genome assemblies for nine Heliconiini, 29 additional reference-assembled genomes, and improve 10 existing assemblies. Altogether, we provide a dataset of annotated genomes for a total of 63 species, including 58 species within the Heliconiini tribe. We use this extensive dataset to generate a robust and dated heliconiine phylogeny, describe major patterns of introgression, explore the evolution of genome architecture, and the genomic basis of key innovations in this enigmatic group, including an assessment of the evolution of putative regulatory regions at the Heliconius stem. Our work illustrates how the increased resolution provided by such dense genomic sampling improves our power to generate and test gene-phenotype hypotheses, and precisely characterize how genomes evolve.

Variation of Clasper Scent Gland Composition of Heliconius Butterflies from a Biodiversity Hotspot.

Male Heliconius butterflies possess two pheromone emitting structures, wing androconia and abdominal clasper scent glands. The composition of the clasper scent gland of males of 17 Heliconius and Eueides species from an overlapping area in Ecuador, comprising three mimicry groups, was investigated by GC/MS. The chemical signal serves as an anti-aphrodisiac signal that is transferred from males to females during mating, indicating the mating status of the female to prevent them from harassment by other males. In addition, the odour may also serve in predator defence. There is potential for convergence driven by mimicry, although, such convergence might be detrimental for species recognition of the butterflies within the mimicry ring, making mating more difficult. More than 500 compounds were detected, consisting of volatile, semi-volatile or non-volatile compounds, including terpenes, fatty acid esters or aromatic compounds. Several novel esters were identified by GC/MS and GC/IR data, microderivatisation and synthesis, including butyl (Z)-3-dodecenoate and other (Z)-3-alkenoates, 3-oxohexyl citronellate and 5-methylhexa-3,5-dienyl (E)-2,3-dihydrofarnesoate. The secretions were found to be species specific, potentially allowing for species differentiation. Statistical analysis of the compounds showed differentiation by phylogenetic clade and species, but not by mimicry group.

Adaptive evolution to the natural and anthropogenic environment in a global invasive crop pest, the cotton bollworm.

The cotton bollworm, Helicoverpa armigera, is set to become the most economically devastating crop pest in the world, threatening food security and biosafety as its range expands across the globe. Key to understanding the eco-evolutionary dynamics of H. armigera, and thus its management, is an understanding of population connectivity and the adaptations that allow the pest to establish in unique environments. We assembled a chromosome-scale reference genome and re-sequenced 503 individuals spanning the species range to delineate global patterns of connectivity, uncovering a previously cryptic population structure. Using a genome-wide association study (GWAS) and cell line expression of major effect loci, we show that adaptive changes in a temperature- and light-sensitive developmental pathway enable facultative diapause and that adaptation of trehalose synthesis and transport underlies cold tolerance in extreme environments. Incorporating extensive pesticide resistance monitoring, we also characterize a suite of novel pesticide and Bt resistance alleles under selection in East China. These findings offer avenues for more effective management strategies and provide insight into how insects adapt to variable climatic conditions and newly colonized environments.

High level of novelty under the hood of convergent evolution.

Little is known about the extent to which species use homologous regulatory architectures to achieve phenotypic convergence. By characterizing chromatin accessibility and gene expression in developing wing tissues, we compared the regulatory architecture of convergence between a pair of mimetic butterfly species. Although a handful of color pattern genes are known to be involved in their convergence, our data suggest that different mutational paths underlie the integration of these genes into wing pattern development. This is supported by a large fraction of accessible chromatin being exclusive to each species, including the de novo lineage-specific evolution of a modular optix enhancer. These findings may be explained by a high level of developmental drift and evolutionary contingency that occurs during the independent evolution of mimicry.

Evolution of physical linkage between loci controlling ecological traits and mating preferences.

Coupling of multiple barriers to gene-flow, such as divergent local adaptation and reproductive isolation, facilitates speciation. However, alleles at loci that contribute to barrier effects can be dissociated by recombination. Models of linkage between diverging alleles often consider elements that reduce recombination, such as chromosomal inversions and alleles that modify recombination rate between existing loci. In contrast, here, we consider the evolution of linkage due to the close proximity of loci on the same chromosome. Examples of such physical linkage exist in several species, but in other cases, strong associations are maintained without physical linkage. We use an individual-based model to study the conditions under which the physical linkage between loci controlling ecological traits and mating preferences might be expected to evolve. We modelled a single locus controlling an ecological trait that acts also as a mating cue. Mating preferences are controlled by multiple loci, formed by mutations that are randomly placed in the "genome", within varying distances from the ecological trait locus, allowing us to examine which genomic architectures spread across the population. Our model reveals that stronger physical linkage is favoured when mating preferences and selection are weaker. Under such conditions mating among divergent phenotypes is more frequent, and matching ecological trait and mating preference alleles are more likely to become dissociated by recombination, favouring the evolution of genetic linkage. While most theoretical studies on clustering of divergent loci focus on how physical linkage influences speciation, we show how physical linkage itself can arise, establishing conditions that can favour speciation.

A butterfly pan-genome reveals that a large amount of structural variation underlies the evolution of chromatin accessibility.

Despite insertions and deletions being the most common structural variants (SVs) found across genomes, not much is known about how much these SVs vary within populations and between closely related species, nor their significance in evolution. To address these questions, we characterized the evolution of indel SVs using genome assemblies of three closely related Heliconius butterfly species. Over the relatively short evolutionary timescales investigated, up to 18.0% of the genome was composed of indels between two haplotypes of an individual Heliconius charithonia butterfly and up to 62.7% included lineage-specific SVs between the genomes of the most distant species (11 Mya). Lineage-specific sequences were mostly characterized as transposable elements (TEs) inserted at random throughout the genome and their overall distribution was similarly affected by linked selection as single nucleotide substitutions. Using chromatin accessibility profiles (i.e., ATAC-seq) of head tissue in caterpillars to identify sequences with potential cis-regulatory function, we found that out of the 31,066 identified differences in chromatin accessibility between species, 30.4% were within lineage-specific SVs and 9.4% were characterized as TE insertions. These TE insertions were localized closer to gene transcription start sites than expected at random and were enriched for sites with significant resemblance to several transcription factor binding sites with known function in neuron development in Drosophila We also identified 24 TE insertions with head-specific chromatin accessibility. Our results show high rates of structural genome evolution that were previously overlooked in comparative genomic studies and suggest a high potential for structural variation to serve as raw material for adaptive evolution.

Repeated genetic adaptation to altitude in two tropical butterflies.

Repeated evolution can provide insight into the mechanisms that facilitate adaptation to novel or changing environments. Here we study adaptation to altitude in two tropical butterflies, Heliconius erato and H. melpomene, which have repeatedly and independently adapted to montane habitats on either side of the Andes. We sequenced 518 whole genomes from altitudinal transects and found many regions differentiated between highland (~ 1200 m) and lowland (~ 200 m) populations. We show repeated genetic differentiation across replicate populations within species, including allopatric comparisons. In contrast, there is little molecular parallelism between the two species. By sampling five close relatives, we find that a large proportion of divergent regions identified within species have arisen from standing variation and putative adaptive introgression from high-altitude specialist species. Taken together our study supports a role for both standing genetic variation and gene flow from independently adapted species in promoting parallel local adaptation to the environment.

Condition dependence in biosynthesized chemical defenses of an aposematic and mimetic Heliconius butterfly.

Aposematic animals advertise their toxicity or unpalatability with bright warning coloration. However, acquiring and maintaining chemical defenses can be energetically costly, and consequent associations with other important traits could shape chemical defense evolution. Here, we have tested whether chemical defenses are involved in energetic trade-offs with other traits, or whether the levels of chemical defenses are condition dependent, by studying associations between biosynthesized cyanogenic toxicity and a suite of key life-history and fitness traits in a Heliconius butterfly under a controlled laboratory setting. Heliconius butterflies are well known for the diversity of their warning color patterns and widespread mimicry and can both sequester the cyanogenic glucosides of their Passiflora host plants and biosynthesize these toxins de novo. We find energetically costly life-history traits to be either unassociated or to show a general positive association with biosynthesized cyanogenic toxicity. More toxic individuals developed faster and had higher mass as adults and a tendency for increased lifespan and fecundity. These results thus indicate that toxicity level of adult butterflies may be dependent on individual condition, influenced by genetic background or earlier conditions, with maternal effects as one strong candidate mechanism. Additionally, toxicity was higher in older individuals, consistent with previous studies indicating accumulation of toxins with age. As toxicity level at death was independent of lifespan, cyanogenic glucoside compounds may have been recycled to release resources relevant for longevity in these long-living butterflies. Understanding the origins and maintenance of variation in defenses is necessary in building a more complete picture of factors shaping the evolution of aposematic and mimetic systems.

Exploitation of an ancestral pheromone biosynthetic pathway contributes to diversification in Heliconius butterflies.

During courtship, male butterflies of many species produce androconial secretions containing male sex pheromones (MSPs) that communicate species identity and affect female choice. MSPs are thus likely candidates as reproductive barriers, yet their role in speciation remains poorly studied. Although Heliconius butterflies are a model system in speciation, their MSPs have not been investigated from a macroevolutionary perspective. We use GC/MS to characterize male androconial secretions in 33 of the 69 species in the Heliconiini tribe. We found these blends to be species-specific, consistent with a role in reproductive isolation. We detected a burst in blend diversification rate at the most speciose genus, Heliconius; a consequence of Heliconius and Eueides species using a fatty acid (FA) metabolic pathway to unlock more complex blends than basal Heliconiini species, whose secretions are dominated by plant-like metabolites. A comparison of 10 sister species pairs demonstrates a striking positive correlation between blend dissimilarity and range overlap, consistent with character displacement or reinforcement in sympatry. These results demonstrate for the first time that MSP diversification can promote reproductive isolation across this group of butterflies, showcasing how implementation of an ancestral trait, the co-option of the FA metabolic pathway for pheromone production, can facilitate rapid speciation.

Alternative splicing as a source of phenotypic diversity.

A major goal of evolutionary genetics is to understand the genetic processes that give rise to phenotypic diversity in multicellular organisms. Alternative splicing generates multiple transcripts from a single gene, enriching the diversity of proteins and phenotypic traits. It is well established that alternative splicing contributes to key innovations over long evolutionary timescales, such as brain development in bilaterians. However, recent developments in long-read sequencing and the generation of high-quality genome assemblies for diverse organisms has facilitated comparisons of splicing profiles between closely related species, providing insights into how alternative splicing evolves over shorter timescales. Although most splicing variants are probably non-functional, alternative splicing is nonetheless emerging as a dynamic, evolutionarily labile process that can facilitate adaptation and contribute to species divergence.

Head and Tail Oxidized Terpenoid Esters from Androconia of Heliconius erato Butterflies.

Heliconius erato is a neotropical butterfly species that is part of a complex mimicry ring, with colorful wing patterns. For intraspecific communication, males use pheromones that are released from two different scent-emitting structures. Scent glands located near the abdominal claspers of males, containing antiaphrodisiac pheromones, release a highly complex mixture of compounds that is transferred to females during mating, rendering them unattractive to other males. On the other hand, androconia, scent-emitting scale areas on the wings of male butterflies, release a structurally more restricted set of compounds that likely serves an aphrodisiac role. We report here on two structurally related compounds that are the major androconial constituents, produced in high amounts and are not volatile due to their high molecular mass. Their structures were established by extensive analysis of mass, infrared, and NMR spectra, as well as microderivatization reactions of the natural extract. After establishing synthetic access, the compounds were unequivocally identified as two unusual head and tail oxidized terpenoids, (4E,8E,12E)-4,8,12-trimethyl-16-oxoheptadeca-4,8,12-trien-1-yl oleate (1) and stearate (2). Although behavioral assays are necessary to fully comprehend their role in the chemical communication of the species, hypotheses for their use by the butterflies are also discussed.

A large deletion at the cortex locus eliminates butterfly wing patterning.

As the genetic basis of natural and domesticated variation has been described in recent years, a number of hotspot genes have been repeatedly identified as the targets of selection, Heliconius butterflies display a spectacular diversity of pattern variants in the wild and the genetic basis of these patterns has been well-described. Here, we sought to identify the mechanism behind an unusual pattern variant that is instead found in captivity, the ivory mutant, in which all scales on both the wings and body become white or yellow. Using a combination of autozygosity mapping and coverage analysis from 37 captive individuals, we identify a 78-kb deletion at the cortex wing patterning locus, a gene which has been associated with wing pattern evolution in H. melpomene and 10 divergent lepidopteran species. This deletion is undetected among 458 wild Heliconius genomes samples, and its dosage explains both homozygous and heterozygous ivory phenotypes found in captivity. The deletion spans a large 5' region of the cortex gene that includes a facultative 5'UTR exon detected in larval wing disk transcriptomes. CRISPR mutagenesis of this exon replicates the wing phenotypes from coding knock-outs of cortex, consistent with a functional role of ivory-deleted elements in establishing scale color fate. Population demographics reveal that the stock giving rise to the ivory mutant has a mixed origin from across the wild range of H. melpomene, and supports a scenario where the ivory mutation occurred after the introduction of cortex haplotypes from Ecuador. Homozygotes for the ivory deletion are inviable while heterozygotes are the targets of artificial selection, joining 40 other examples of allelic variants that provide heterozygous advantage in animal populations under artificial selection by fanciers and breeders. Finally, our results highlight the promise of autozygosity and association mapping for identifying the genetic basis of aberrant mutations in captive insect populations.